Asparagine-linked sugar chains (also abbreviated as “N-linked sugar chains”) are composed of a structure in which a sugar chain is bound to an amide nitrogen atom of the side chain of asparagine (Asn), and may have a wide range of structures depending on such factors as the types and arrangement of monosaccharides composing the sugar chain or the presence or absence of branching. N-linked sugar chains are intimately involved in various biological functions, such as immune response regulation, cell growth, malignant transformation or metastasis of cancer cells, through interaction with other molecules such as proteins or lipids, while also contributing to stability of proteins in the body. Since the functions of these N-linked sugar chains can be expected to lead to applications in pharmaceuticals for diagnosis and treatment, analyses are being conducted on the pharmacokinetics of these N-linked sugar chains in the body. Since the interaction between N-linked sugar chains and proteins is mainly dependent on sugar chain structure, methods used to analyze the functions of N-linked sugar chains consist of administering a glycoprotein, obtained by binding an N-linked sugar chain having a specific sugar chain structure to a protein such as albumin, to an animal and analyzing parameters such as pharmacokinetics or the presence or absence of accumulation in tissue (see, for example, Non-Patent Document 1 or 2). In addition, N-linked sugar chains are also investigated non-invasively by further introducing a fluorescent substance into a glycoprotein having an N-linked sugar chain of a specific structure and analyzing the pharmacokinetics of the glycoprotein in the body of the animal by, for example, bioimaging (see, for example, Non-Patent Document 3).
Although the interaction between a single sugar chain and a protein is weak, a strong interaction is demonstrated when multiple sugar chains accumulate (sugar chain clustering effect). Consequently, in order to obtain this sugar chain clustering effect, complexes obtained by binding as many N-linked sugar chains as possible to a single protein molecule are preferable for use as glycoproteins used for functional analysis of N-linked sugar chains.
The inventors of the present invention previously reported that pharmacokinetics were analyzed by introducing 4 to 16 molecules of N-linked sugar chains per protein molecule into a polylysine skeleton and further synthesizing a sugar chain cluster in which the terminals thereof were modified with a fluorescent substance (see Non-Patent Document 4). Since sugar chains are bulky and have numerous hydroxy groups, it has conventionally been extremely difficult to increase the number of sugar chain molecules bound to a single protein molecule.